Chromothripsis Is Enriched at Relapse of Childhood B-Cell Precursor Acute Lymphoblastic Leukemia and Cooccurs with Loss of Function of TP53 and RB1 Genes

Introduction: Chromothripsis (cth) is a form of genomic instability characterized by the simultaneous fragmentation of one or several chromosomal regions, followed by the loss of DNA fragments and their random reassembly. This phenomenon can lead to the loss of tumor suppressor gene functions, oncogenes amplification, and the formation of cancer-promoting gene fusions. In B-cell precursor acute lymphoblastic leukemia (BCP-ALL), chromothripsis has been described as an intrinsic feature accompanying intrachromosomal amplification of chromosome 21 (iAmp21).

Aim of the study: The study aimed to assess the frequency and prognostic significance of chromothripsis in the context of somatic genotype and germline aberrations.

Materials and Methods: The study group comprised n=567 pediatric patients with newly diagnosed BCP-ALL treated with the AIEOP-BFM 2017 protocol and n=375 children with leukemia relapse who received therapy according to IntRE-ALL 2010 protocol. Analysis of copy number aberrations and the occurrence of cth was performed using high-resolution microarrays in n=446 BCP-ALL and n=181 relapses, respectively. Sequence variants and gene fusions were identified using RNA sequencing. To detect cth-induced both balanced and unbalanced structural variants in n=14 cth positive ALL samples we used optical genome mapping (OGM) .

Results: The incidence of chromothripsis was almost two and half times higher in BCP-ALL relapses as compared to the primary leukemia diagnoses [n=17/446 (3.96%) and n=17/181 (9.39%); p=0.005]. There were no statistically significant differences in cth positive BCP-ALL compared to negative ALL in the probability of 5-year overall (0.71 vs. 0.91; p=0.11), relapse-free (0.68 vs. 0.82; p=0.51), and event-free survival (0.63 vs. 0.78; p=0.46). However, the median follow-up time was short: 3.2 (2.41-4.08) in the cth-positive group vs. 3.23 (2.41-4.22) in cth-negative patients.

In 5/12 (41.67%) patients with paired samples from both timepoints, cth was observed exclusively at BCP-ALL relapse. Cth recurrently affected chromosomes: chr. 21 (n=9), chr. 7 and chr. 9 (n=4), and in n=5/31 (16.13%) patients cth involved more than one chromosome. Chromothripsis was present across different molecular entities of BCP-ALL including: [B-other (n=7); iAmp21 (n=7); ETV6::RUNX1 (n=5); Hyperdiploidy (n=4); KMT2Ar, PAX5r, Ph-like - (n=2); TP53mut, Hypodiploidy, dic(9;20), and MEF2Dr – (n=1)]. However, the most common co-occurring aberrations involved tumor suppressor genes TP53 (n=9) and RB1 (n=7), which were identified in almost half of cth positive BCP-ALL samples (n=14/31; 45.16 %). Cth generated cancer-promoting defects including deletions of IKZF1 and RB1 genes. Optical genome mapping was performed in 14/31 (45.16%) cth positive cases and revealed several gene fusions generated by chromothripsis with a median number per sample reaching 5.5 (4.25-10). Fusions comprised IL7::IGH, DNMT1::POLR2E, and in one case complex translocation affecting: PAX5, AUTS2, and CBFA2T2 genes was detected. Additionally, in n=3/31 patients (9.68%) inherited germline predisposition to cancer was identified: Li-Fraumeni syndrome (n=2) and Nijmegen syndrome (n=1), respectively.

Conclusions: The incidence of chromothripsis was increased in ALL relapse samples. Almost half of the cth-postive BCP-ALL cases showed loss of function of the tumor suppressor genes TP53 and RB1. Chromothripsis by generating massive structural rearrangements contributes to clonal evolution of leukemia and may possibly promote disease recurrence.